Universal CAR T cells targeted to HER2 with a biotin-trastuzumab soluble linker penetrate spheroids and large tumor xenografts that are inherently resistant to trastuzumab mediated ADCC.

CAR T cell therapies face challenges in combating solid tumors due to their single-target approach, which becomes ineffective if the targeted antigen is absent or lost. Universal CAR T cells (UniCAR Ts) provide a promising solution by utilizing molecular tags (linkers), such as biotin conjugated to monoclonal antibodies, enabling them to target a variety of tumor antigens. Recently, we showed that conventional CAR T cells could penetrate the extracellular matrix (ECM) of ADCC-resistant tumors, which forms a barrier to therapeutic antibodies. This finding led us to investigate whether UniCAR T cells, targeted by soluble antibody-derived linkers, could similarly tackle ADCC-resistant tumors where ECM restricts antibody penetration. We engineered UniCAR T cells by incorporating a biotin-binding monomeric streptavidin 2 (mSA2) domain for targeting HER2 via biotinylated trastuzumab (BT). The activation and cytotoxicity of UniCAR T cells in the presence or absence of BT were evaluated in conventional immunoassays. A 3D spheroid coculture was set up to test the capability of UniCAR Ts to access ECM-masked HER2+ cells. For in vivo analysis, we utilized a HER2+ xenograft model in which intravenously administered UniCAR T cells were supplemented with intraperitoneal BT treatments. In vitro, BT-guided UniCAR T cells showed effective activation and distinct anti-tumor response. Upon target recognition, IFNγ secretion correlated with BT concentration. In the presence of BT, UniCAR T cells effectively penetrated HER2+ spheroids and induced cell death in their core regions. In vivo, upon intravenous administration of UniCAR Ts, circulating BT linkers immediately engaged the mSA2 domain and directed effector cells to the HER2+ tumors. However, these co-treated mice died early, possibly due to the lung infiltration of UniCAR T cells that could recognize both native biotin and HER2. Our results suggest that UniCAR T cells guided with soluble linkers present a viable alternative to conventional CAR T cells, especially for patients resistant to antibody therapy and those with solid tumors exhibiting high antigenic variability. Critical to their success, however, is the choice of an appropriate binding domain for the CAR and the corresponding soluble linker, ensuring both efficacy and safety in therapeutic applications.

Engineering Saccharomyces cerevisiae for fast vitamin-independent aerobic growth.

Chemically defined media for cultivation of Saccharomyces cerevisiae strains are commonly supplemented with a mixture of multiple Class-B vitamins, whose omission leads to strongly reduced growth rates. Fast growth without vitamin supplementation is interesting for industrial applications, as it reduces costs and complexity of medium preparation and may decrease susceptibility to contamination by auxotrophic microbes. In this study, suboptimal growth rates of S. cerevisiae CEN.PK113-7D in the absence of pantothenic acid, para-aminobenzoic acid (pABA), pyridoxine, inositol and/or biotin were corrected by single or combined overexpression of ScFMS1, ScABZ1/ScABZ2, ScSNZ1/ScSNO1, ScINO1 and Cyberlindnera fabianii BIO1, respectively. Several strategies were explored to improve growth of S. cerevisiae CEN.PK113-7D in thiamine-free medium. Overexpression of ScTHI4 and/or ScTHI5 enabled thiamine-independent growth at 83% of the maximum specific growth rate of the reference strain in vitamin-supplemented medium. Combined overexpression of seven native S. cerevisiae genes and CfBIO1 enabled a maximum specific growth rate of 0.33 ± 0.01 h-1 in vitamin-free synthetic medium. This growth rate was only 17 % lower than that of a congenic reference strain in vitamin-supplemented medium. Physiological parameters of the engineered vitamin-independent strain in aerobic glucose-limited chemostat cultures (dilution rate 0.10 h-1) grown on vitamin-free synthetic medium were similar to those of similar cultures of the parental strain grown on vitamin-supplemented medium. Transcriptome analysis revealed only few differences in gene expression between these cultures, which primarily involved genes with roles in Class-B vitamin metabolism. These results pave the way for development of fast-growing vitamin-independent industrial strains of S. cerevisiae.

Optimisation of denaturing ion pair reversed phase HPLC for the purification of ssDNA in SELEX.

Aptamers have shown great promise as oligonucleotide-based affinity ligands for various medicinal and industrial applications. A critical step in the production of DNA aptamers via selective enhancement of ligands by exponential enrichment (SELEX) is the generation of ssDNA from dsDNA. There are a number of caveats associated with current methods for ssDNA generation, which can lower success rates of SELEX experiments. They often result in low yields thereby decreasing diversity or fail to eliminate parasitic PCR by-products leading to accumulation of by-products from round to round. Both contribute to the failure of SELEX protocols and therefore potentially limit the impact of aptamers compared to their peptide-based antibody counterparts. We have developed a novel method using ion pair reversed phase HPLC (IP RP HPLC) employed under denaturing conditions for the ssDNA re-generation stage of SELEX following PCR. We have utilised a range of 5' chemical modifications on PCR primers to amplify PCR fragments prior to separation and purification of the DNA strands using denaturing IP RP HPLC. We have optimised mobile phases to enable complete denaturation of the dsDNA at moderate temperatures that circumvents the requirement of high temperatures and results in separation of the ssDNA based on differences in their hydrophobicity. Validation of the ssDNA isolation and purity assessment was performed by interfacing the IP RP HPLC with mass spectrometry and fluorescence-based detection. The results show that using a 5' Texas Red modification on the reverse primer in the PCR stage enabled purification of the ssDNA from its complimentary strand via IP RP HPLC under denaturing conditions. Additionally, we have confirmed the purity of the ssDNA generated as well as the complete denaturation of the PCR product via the use of mass-spectrometry and fluorescence analysis therefore proving the selective elimination of PCR by-products and the unwanted complementary strand. Following lyophilisation, ssDNA yields of up to 80% were obtained. In comparison the streptavidin biotin affinity chromatography also generates pure ssDNA with a yield of 55%. The application of this method to rapidly generate and purify ssDNA of the correct size, offers the opportunity to improve the development of new aptamers via SELEX.

Strictosamide ameliorates LPS-induced acute lung injury by targeting ERK2 and mediating NF-κB signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Acute lung injury (ALI) ranks among the deadliest pulmonary diseases, significantly impacting mortality and morbidity. Presently, the primary treatment for ALI involves supportive therapy; however, its efficacy remains unsatisfactory. Strictosamide (STR), an indole alkaloid found in the Chinese herbal medicine Nauclea officinalis (Pierre ex Pit.) Merr. & Chun (Wutan), has been found to exhibit numerous pharmacological properties, particularly anti-inflammatory effects. AIM OF THE STUDY: This study aimes to systematically identify and validate the specific binding proteins targeted by STR and elucidate its anti-inflammatory mechanism in lipopolysaccharide (LPS)-induced ALI. MATERIALS AND METHODS: Biotin chemical modification, protein microarray analysis and network pharmacology were conducted to screen for potential STR-binding proteins. The binding affinity was assessed through surface plasmon resonance (SPR), cellular thermal shift assay (CETSA) and molecular docking, and the anti-inflammatory mechanism of STR in ALI treatment was assessed through in vivo and in vitro experiments. RESULTS: Biotin chemical modification, protein microarray and network pharmacology identified extracellular-signal-regulated kinase 2 (ERK2) as the most important binding proteins among 276 candidate STR-interacting proteins and nuclear factor-kappaB (NF-κB) pathway was one of the main inflammatory signal transduction pathways. Using SPR, CETSA, and molecular docking, we confirmed STR's affinity for ERK2. In vitro and in vivo experiments demonstrated that STR mitigated inflammation by targeting ERK2 to modulate the NF-κB signaling pathway in LPS-induced ALI. CONCLUSIONS: Our findings indicate that STR can inhibit the NF-κB signaling pathway to attenuate LPS-induced inflammation by targeting ERK2 and decreasing phosphorylation of ERK2, which could be a novel strategy for treating ALI.

Crocin Ameliorates Diabetic Nephropathy through Regulating Metabolism, CYP4A11/PPARγ, and TGF-ß/Smad Pathways in Mice.

INTRODUCTION: Crocin is one of the main components of Crocus sativus L. and can alleviate oxidative stress and inflammation in diabetic nephropathy (DN). However, the specific mechanism by which crocin treats DN still needs to be further elucidated. METHOD: In the present study, a mouse model of DN was first established to investigate the therapeutic effect of crocin on DN mice. Subsequently, non-targeted metabolomics techniques were used to analyze the mechanisms of action of crocin in the treatment of DN. The effects of crocin on CYP4A11/PPARγ and TGF-ß/Smad pathway were also investigated. RESULT: Results showed that crocin exhibited significant therapeutic and anti-inflammatory, and anti-oxidative effects on DN mice. In addition, the non-targeted metabolomics results indicated that crocin treatment affected several metabolites in kidney. These metabolites were mainly associated with biotin metabolism, riboflavin metabolism, and arachidonic acid metabolism. Furthermore, crocin treatment upregulated the decreased levels of CYP4A11 and phosphorylated PPARγ, and reduced the increased levels of TGF-ß1 and phosphorylated Smad2/3 in the kidneys of DN mice. CONCLUSION: In conclusion, our study validated the considerable therapeutic, anti-inflammatory, and antioxidative impacts of crocin on DN mice. The mechanism of crocin treatment may be related to the regulation of biotin riboflavin and arachidonic acid metabolism, the activation of CYP4A11/PPARγ pathway, and the inhibition of TGF-ß/Smad pathway in the kidney.

Neuroimaging Features of Biotinidase Deficiency.

Biotinidase deficiency is an autosomal recessive condition caused by pathogenic variants in the BTD gene. Resultant deficiency of free biotin leads to impaired activity of the enzyme carboxylase and related neurologic, dermatologic, and ocular symptoms. Many of these are reversible on treatment, but early recognition and commencement of biotin supplementation are critical. This practice is especially important in countries where routine neonatal screening for biotinidase deficiency is not performed. In this report comprising 14 patients from multiple centers, we demonstrate the MR imaging patterns of this disorder at various age groups. Knowledge of these patterns in the appropriate clinical context will help guide early diagnosis of this treatable metabolic disorder.

Huntington disease oligodendrocyte maturation deficits revealed by single-nucleus RNAseq are rescued by thiamine-biotin supplementation.

The complexity of affected brain regions and cell types is a challenge for Huntington's disease (HD) treatment. Here we use single nucleus RNA sequencing to investigate molecular pathology in the cortex and striatum from R6/2 mice and human HD post-mortem tissue. We identify cell type-specific and -agnostic signatures suggesting oligodendrocytes (OLs) and oligodendrocyte precursors (OPCs) are arrested in intermediate maturation states. OL-lineage regulators OLIG1 and OLIG2 are negatively correlated with CAG length in human OPCs, and ATACseq analysis of HD mouse NeuN-negative cells shows decreased accessibility regulated by OL maturation genes. The data implicates glucose and lipid metabolism in abnormal cell maturation and identify PRKCE and Thiamine Pyrophosphokinase 1 (TPK1) as central genes. Thiamine/biotin treatment of R6/1 HD mice to compensate for TPK1 dysregulation restores OL maturation and rescues neuronal pathology. Our insights into HD OL pathology spans multiple brain regions and link OL maturation deficits to abnormal thiamine metabolism.

Dietary High-Dose Biotin Intake Activates Fat Oxidation and Hepatic Carnitine Palmitoyltransferase in Rat.

This study investigated the effects of dietary high-dose biotin intake on fat oxidation in rats using respiratory gas analysis, and evaluated fatty-acid oxidation-related enzyme activities and gene expressions in the liver. Five-week-old male Sprague-Dawley rats were fed a control diet and three biotin-supplemented diets (additive biotin concentration: 0.05%, 0.10%, and 0.20% of diet) for 3 wk. In 2 wk, fat oxidation in the 0.20% biotin-supplemented diet group was higher than that in the 0.05% biotin-supplemented diet group; however, the energy expenditure and carbohydrate oxidation were unchanged between the dietary groups. At the end of 3 wk, body weight and epididymal white adipose tissue weight reduced in the 0.20% biotin diet group, and hepatic triglyceride levels tended to decrease. Additionally, increased plasma adiponectin concentration and hepatic mitochondrial carnitine palmitoyltransferase activity as well as decreased hepatic acetyl-CoA carboxylase 2 gene expression were observed in the 0.20% biotin-supplemented diet group compared with those in the control group. These results provide strong evidence that dietary high-dose biotin intake activated fat oxidation due to the increase in hepatic ß-oxidation, which may contribute to the decrease in hepatic triglyceride concentration and white adipose tissue weight.

Metabolic adaptation to vitamin auxotrophy by leaf-associated bacteria.

Auxotrophs are unable to synthesize all the metabolites essential for their metabolism and rely on others to provide them. They have been intensively studied in laboratory-generated and -evolved mutants, but emergent adaptation mechanisms to auxotrophy have not been systematically addressed. Here, we investigated auxotrophies in bacteria isolated from Arabidopsis thaliana leaves and found that up to half of the strains have auxotrophic requirements for biotin, niacin, pantothenate and/or thiamine. We then explored the genetic basis of auxotrophy as well as traits that co-occurred with vitamin auxotrophy. We found that auxotrophic strains generally stored coenzymes with the capacity to grow exponentially for 1-3 doublings without vitamin supplementation; however, the highest observed storage was for biotin, which allowed for 9 doublings in one strain. In co-culture experiments, we demonstrated vitamin supply to auxotrophs, and found that auxotrophic strains maintained higher species richness than prototrophs upon external supplementation with vitamins. Extension of a consumer-resource model predicted that auxotrophs can utilize carbon compounds provided by other organisms, suggesting that auxotrophic strains benefit from metabolic by-products beyond vitamins.

Studying the ubiquitin code through biotin-based labelling methods.

Post-translational modifications of cellular substrates by members of the ubiquitin (Ub) and ubiquitin-like (UbL) family are crucial for regulating protein homeostasis in organisms. The term "ubiquitin code" encapsulates how this diverse family of modifications, via adding single UbLs or different types of UbL chains, leads to specific fates for substrates. Cancer, neurodegeneration and other conditions are sometimes linked to underlying errors in this code. Studying these modifications in cells is particularly challenging since they are usually transient, scarce, and compartment-specific. Advances in the use of biotin-based methods to label modified proteins, as well as their proximally-located interactors, facilitate isolation and identification of substrates, modification sites, and the enzymes responsible for writing and erasing these modifications, as well as factors recruited as a consequence of the substrate being modified. In this review, we discuss site-specific and proximity biotinylation approaches being currently applied for studying modifications by UbLs, highlighting the pros and cons, with mention of complementary methods when possible. Future improvements may come from bioengineering and chemical biology but even now, biotin-based technology is uncovering new substrates and regulators, expanding potential therapeutic targets to manipulate the Ub code.

Effect of chronic alcohol exposure on gut vitamin B7 uptake: involvement of epigenetic mechanisms and effect of alcohol metabolites.

Vitamin B7 (biotin) is essential for normal health and its deficiency/suboptimal levels occur in a variety of conditions including chronic alcoholism. Mammals, including humans, obtain biotin from diet and gut-microbiota via absorption along the intestinal tract. The absorption process is carrier mediated and involves the sodium-dependent multivitamin transporter (SMVT; SLC5A6). We have previously shown that chronic alcohol exposure significantly inhibits intestinal/colonic biotin uptake via suppression of Slc5a6 transcription in animal and cell line models. However, little is known about the transcriptional/epigenetic factors that mediate this suppression. In addition, the effect of alcohol metabolites (generated via alcohol metabolism by gut microbiota and host tissues) on biotin uptake is still unknown. To address these questions, we first demonstrated that chronic alcohol exposure inhibits small intestinal and colonic biotin uptake and SMVT expression in human differentiated enteroid and colonoid monolayers. We then showed that chronic alcohol exposures of both, Caco-2 cells and mice, are associated with a significant suppression in expression of the nuclear factor KLF-4 (needed for Slc5a6 promoter activity), as well as with epigenetic alterations (histone modifications). We also found that chronic exposure of NCM460 human colonic epithelial cells as well as human differentiated colonoid monolayers, to alcohol metabolites (acetaldehyde, ethyl palmitate, ethyl oleate) significantly inhibited biotin uptake and SMVT expression. These findings shed light onto the molecular/epigenetic mechanisms that mediate the inhibitory effect of chronic alcohol exposure on intestinal biotin uptake. They further show that alcohol metabolites are also capable of inhibiting biotin uptake in the gut.NEW & NOTEWORTHY Using complementary models, including human differentiated enteroid and colonoid monolayers, this study shows the involvement of molecular and epigenetic mechanisms in mediating the inhibitory effect of chronic alcohol exposure on biotin uptake along the intestinal tract. The study also shows that alcohol metabolites (generated by gut microbiota and host tissues) cause inhibition in gut biotin uptake.

Effect of Biotin on Cardiac Troponin I and High Sensitivity Cardiac Troponin I Assays on Vista 1500 and ADVIA Centaur Analyzer.

OBJECTIVE: Biotin interferes with biotinylated antibody based immunoassays. We investigated effect of biotin on conventional troponin I and two high sensitivity troponin I assays, all manufactured by Siemens. MATERIALS AND METHODS: One high sensitivity troponin I assay (TNIH-1) was run using ADVIA Centaur analyzer. The second high sensitivity troponin I assay (TNIH-2) as well as conventional troponin I assay (CTNI) were run using Dimension Vista 1500 analyzer. We analyzed 25 specimens using CTNI, TNIH-1 and TNIH-2 assays for comparison of these assays. Moreover, serum pools prepared from additional specimens containing various amounts of troponin I were further supplemented with biotin to achieve biotin concentrations between 50 and 1000 ng/mL followed by reanalysis using CTNI, TNIH-1 and TNIH-2 assays. RESULTS: Although both high sensitivity troponin I assays correlated well, there was a significant positive bias with TNIH-2. We observed no significant negative biotin interference at a level up to 250 ng/mL. Highest observed negative bias was 29.7%. CONCLUSIONS: All three troponin I assays were free from biotin interferences up to a biotin concentration of 250 ng/mL.

High-dose biotin restores redox balance, energy and lipid homeostasis, and axonal health in a model of adrenoleukodystrophy.

Biotin is an essential cofactor for carboxylases that regulates the energy metabolism. Recently, high-dose pharmaceutical-grade biotin (MD1003) was shown to improve clinical parameters in a subset of patients with chronic progressive multiple sclerosis. To gain insight into the mechanisms of action, we investigated the efficacy of high-dose biotin in a genetic model of chronic axonopathy caused by oxidative damage and bioenergetic failure, the Abcd1- mouse model of adrenomyeloneuropathy. High-dose biotin restored redox homeostasis driven by NRF-2, mitochondria biogenesis and ATP levels, and reversed axonal demise and locomotor impairment. Moreover, we uncovered a concerted dysregulation of the transcriptional program for lipid synthesis and degradation in the spinal cord likely driven by aberrant SREBP-1c/mTORC1signaling. This resulted in increased triglyceride levels and lipid droplets in motor neurons. High-dose biotin normalized the hyperactivation of mTORC1, thus restoring lipid homeostasis. These results shed light into the mechanism of action of high-dose biotin of relevance for neurodegenerative and metabolic disorders.

Exploring the additive bio-agent impacts upon ectoine production by Halomonas salina DSM5928T using corn steep liquor and soybean hydrolysate as nutrient supplement.

Ectoine production using inexpensive and renewable biomass resources has attracted great interest among the researchers due to the low yields of ectoine in current fermentation approaches that complicate the large-scale production of ectoine. In this study, ectoine was produced from corn steep liquor (CSL) and soybean hydrolysate (SH) in replacement to yeast extract as the nitrogen sources for the fermentation process. To enhance the bacterial growth and ectoine production, biotin was added to the Halomonas salina fermentation media. In addition, the effects addition of surfactants such as Tween 80 and saponin on the ectoine production were also investigated. Results showed that both the CSL and SH can be used as the nitrogen source substitutes in the fermentation media. Higher amount of ectoine (1781.9 mg L-1) was produced in shake flask culture with SH-containing media as compared to CSL-containing media. A total of 2537.0 mg L-1 of ectoine was produced at pH 7 when SH-containing media was applied in the 2 L batch fermentation. Moreover, highest amount of ectoine (1802.0 mg L-1) was recorded in the SH-containing shake flask culture with addition of 0.2 µm mL-1 biotin. This study demonstrated the efficacy of industrial waste as the nutrient supplement for the fermentation of ectoine production.

Enantioselective Hydroxylation of Benzylic C(sp3)-H Bonds by an Artificial Iron Hydroxylase Based on the Biotin-Streptavidin Technology.

The selective hydroxylation of C-H bonds is of great interest to the synthetic community. Both homogeneous catalysts and enzymes offer complementary means to tackle this challenge. Herein, we show that biotinylated Fe(TAML)-complexes (TAML = Tetra Amido Macrocyclic Ligand) can be used as cofactors for incorporation into streptavidin to assemble artificial hydroxylases. Chemo-genetic optimization of both cofactor and streptavidin allowed optimizing the performance of the hydroxylase. Using H2O2 as oxidant, up to ∼300 turnovers for the oxidation of benzylic C-H bonds were obtained. Upgrading the ee was achieved by kinetic resolution of the resulting benzylic alcohol to afford up to >98% ee for (R)-tetralol. X-ray analysis of artificial hydroxylases highlights critical details of the second coordination sphere around the Fe(TAML) cofactor.

Exploiting the Diversity of Saccharomycotina Yeasts To Engineer Biotin-Independent Growth of Saccharomyces cerevisiae.

Biotin, an important cofactor for carboxylases, is essential for all kingdoms of life. Since native biotin synthesis does not always suffice for fast growth and product formation, microbial cultivation in research and industry often requires supplementation of biotin. De novo biotin biosynthesis in yeasts is not fully understood, which hinders attempts to optimize the pathway in these industrially relevant microorganisms. Previous work based on laboratory evolution of Saccharomyces cerevisiae for biotin prototrophy identified Bio1, whose catalytic function remains unresolved, as a bottleneck in biotin synthesis. This study aimed at eliminating this bottleneck in the S. cerevisiae laboratory strain CEN.PK113-7D. A screening of 35 Saccharomycotina yeasts identified six species that grew fast without biotin supplementation. Overexpression of the S. cerevisiaeBIO1 (ScBIO1) ortholog isolated from one of these biotin prototrophs, Cyberlindnera fabianii, enabled fast growth of strain CEN.PK113-7D in biotin-free medium. Similar results were obtained by single overexpression of C. fabianii BIO1 (CfBIO1) in other laboratory and industrial S. cerevisiae strains. However, biotin prototrophy was restricted to aerobic conditions, probably reflecting the involvement of oxygen in the reaction catalyzed by the putative oxidoreductase CfBio1. In aerobic cultures on biotin-free medium, S. cerevisiae strains expressing CfBio1 showed a decreased susceptibility to contamination by biotin-auxotrophic S. cerevisiae This study illustrates how the vast Saccharomycotina genomic resources may be used to improve physiological characteristics of industrially relevant S. cerevisiaeIMPORTANCE The reported metabolic engineering strategy to enable optimal growth in the absence of biotin is of direct relevance for large-scale industrial applications of S. cerevisiae Important benefits of biotin prototrophy include cost reduction during the preparation of chemically defined industrial growth media as well as a lower susceptibility of biotin-prototrophic strains to contamination by auxotrophic microorganisms. The observed oxygen dependency of biotin synthesis by the engineered strains is relevant for further studies on the elucidation of fungal biotin biosynthesis pathways.

Metabolomic Analysis of Liver from Dietary Biotin Deficient Mice.

Biotin is a water-soluble B-complex vitamin that functions as a cofactor of five carboxylases. Because biotin-dependent carboxylases catalyze indispensable cellular metabolic functions, biotin deficiency is considered to be involved in various pathological conditions. Moreover, biotin supplementation shows pharmacological effects in vivo. However, the precise mechanisms by which biotin deficiency induces pathological conditions remain unclear. Although abnormal metabolites are used as indicators for biotin deficiency, few comprehensive analyses of total metabolites have been reported. In this study, we analyzed the metabolomic profiles of liver extracts prepared from biotin-sufficient (BS) and -deficient (BD) mice. Thirteen of 126 metabolites showed significantly different concentrations between liver extracts from BD and BS mice. The concentrations of 5 essential amino acids, Met, Val, Thr, Ile, and Leu, and 2 conditionally essential amino acids, Cys and Tyr were significantly lower in BD mice than in BS mice. Among these, the concentrations of sulfur-containing amino acids, Cys and Met, were more than 1.5-fold lower in BD mice. The concentrations of Met metabolites, such as S-adenosylmethionine and S-adenosylhomocysteine were not significantly different between the two groups. The concentrations of glutathione and its reaction intermediates γ-Glu-Cys tendency to be lower in BD mice. The present study revealed that biotin deficiency induces an abnormal amino acids composition, especially among sulfur-containing amino acids and provide important information on the effect of biotin as a pharmacological agent.

A new bivalent fluorescent fusion protein for differential Cu(II) and Zn(II) ion detection in aqueous solution.

Simple and easy to engineer metal-sensing molecules that are capable of differentiating metal ions and producing metal-specific signals are highly desirable. Metal ions affect the thermal stability of proteins by increasing or decreasing their resistance to unfolding. This work illustrates a new strategy for designing bivalent fluorescent fusion proteins capable of differentiating metal ions in solution through their distinct effects on a protein's thermal stability. A new dual purpose metal sensor was developed consisting of biotin protein ligase (BirA) from B. pseudomallei (Bp) fused to green fluorescent protein (GFP). When coupled with differential scanning fluorimetry of GFP-tagged proteins (DSF-GTP) for signal-transduction detection, Bp BirA-GFP yields distinct protein unfolding signatures with Zn(II) and Cu(II) ions in aqueous solutions. The limit of detection of the system is ∼1 µM for both metal species. The system can be used in a variety of high-throughput assay formats including for the screening of metal-binding proteins and chelators. Bp BirA-GFP has also the additional benefit of being useful in Cu(II) ion field-testing applications through simple visual observation of a temperature-dependent loss of fluorescence. Bp BirA-GFP is the first example of a 2protein-based dual purpose Cu(II) and Zn(II) ion sensor compatible with two different yet complementary signal-transduction detection systems.

Astroglial biotin deprivation under endoplasmic reticulum stress uncouples BCAA-mTORC1 role in lipid synthesis to prolong autophagy inhibition in the aging brain.

Autophagy delays the onset of endoplasmic reticulum (ER) stress by recycling cellular debris. However, the cues that elicit autophagy under the emergence of ER stress and their dysregulation during aging remains obscure. Amino acids, notably branched-chain amino acids (BCAA), get accumulated in the cells once protein synthesis is inhibited by ER stress. The BCAA mimic satiety to inhibit autophagy via mechanistic targets of rapamycin complex 1 (mTORC1) activation and, in contrast, their catabolism supplements de novo lipogenesis for the formation of autophagosome membranes. Thus promoting BCAA utilization is hypothesized to induce autophagy to alleviate ER stress. Nevertheless, except protein synthesis, the rest of BCAA utilization and lipogenesis depends on the co-enzyme biotin. Hence, the levels of biotinylated carboxylases and lipids were assessed in the aging brain of Wistar rats. Despite the increased levels of biotinylated carboxylases and lipids, the aging brain accumulates BCAA. Since astrocytes are the primary site of BCAA and lipid metabolism and the increased expression of glial fibrillary acidic protein (GFAP) denotes astroglial ER stress, co-localization studies were performed to determine the extent of biotinylation in GFAP positive cells. Although total biotin intensity was higher in aged brain slices, the astrocytes specific decrease in biotinylation is attributed to BCAA accumulation, mTORC1 overactivation, autophagy inhibition, and ER stress in the aging brain. The ER stress in primary astrocytes using tunicamycin also mimic the in vivo phenotype. Biotin supplementation ameliorated the changes observed in vitro, corroborating the significance of astrocytes biotin availability to promote autophagy under ER stress in aging.